6.2: Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.

Many Proteins Orchestrate Replication at the Origin

Eukaryotic replication follows many of the same principles as prokaryotic DNA replication, but because the genome is much larger and the chromosomes are linear rather than circular, the process requires more proteins and has a few key differences. Replication occurs simultaneously at multiple origins of replication along each chromosome. Initiator proteins recognize and bind to the origin, recruiting helicase to unwind the DNA double helix. At each point of origin, two replication forks form. Primase then adds short RNA primers to the single strands of DNA, which serve as a starting point for DNA polymerase to bind and begin copying the sequence. DNA can only be synthesized in the 5’ to 3’ direction, so replication of both strands from a single replication fork proceeds in two different directions. The leading strand is synthesized continuously, while the lagging strand is synthesized in short stretches 100-200 base pairs in length, called Okazaki fragments. Once the bulk of replication is complete, RNase enzymes remove the RNA primers and DNA ligase joins any gaps in the new strand.

Dividing the Work of Replication among Polymerases

The workload of copying DNA in eukaryotes is divided among multiple different types of DNA polymerase enzymes. Major families of DNA polymerases across all organisms are categorized by the similarity of their protein structures and amino acid sequences. The first families to be discovered were termed A, B, C, and X, with families Y and D identified later. Family B polymerases in eukaryotes include Pol α, which also functions as a primase at the replication fork, and Pol δ and ε, the enzymes that do most of the work of DNA replication on the leading and lagging strands of the template, respectively. Other DNA polymerases are responsible for such tasks as repairing DNA damage,copying mitochondrial and plastid DNA, and filling in gaps in the DNA sequence on the lagging strand after the RNA primers are removed.

Telomeres Protect the Ends of the Chromosomes from Degradation

Because eukaryotic chromosomes are linear, they are susceptible to degradation at the ends. To protect important genetic information from damage, the ends of chromosomes contain many non-coding repeats of highly conserved G-rich DNA: the telomeres. A short single-stranded 3’ overhang at each end of the chromosome interacts with specialized proteins, which stabilizes the chromosome within the nucleus. Because of the manner in which the lagging strand is synthesized, a small amount of the telomeric DNA cannot be replicated with each cell division. As a result, the telomeres gradually get shorter over the course of many cell cycles and they can be measured as a marker of cellular aging. Certain populations of cells, such as germ cells and stem cells, express telomerase, an enzyme that lengthens the telomeres, allowing the cell to undergo more cell cycles before the telomeres shorten.

Most prokaryotic factors utilized during replication have equivalents that play similar roles in eukaryotic DNA duplication.

This process initiates at an origin of replication, to which a recognition complex binds. Helicase is then attracted to the site and separates the strands of DNA, generating a bubble with two forks.

Primase also arrives and generates RNA primers, which, as helicase moves, DNA polymerase elongates with new DNA. As in prokaryotes, the newly-formed leading strand grows continuously, following the replication fork.

Conversely, the lagging strand is manufactured in small Okazaki fragments, traveling opposite the fork.

Due to multiple factors, the DNA template used to generate the leading strand in 1/2 of this structure creates the lagging strand in the other.

Interestingly, various origins of replication exist on a linear eukaryotic chromosome, and replication terminates when their associated spheres coalesce. Primers are then eliminated via enzymes like RNAse and swapped for DNA. Afterwards, DNA ligase attaches any segments.

However, when the end primer disappears from the lagging strand, the space remains empty, and there is an uncopied stretch of DNA template abutting it. To combat this, an enzyme called telomerase affixes to the overhanging region and elongates it with a non-coding DNA sequence.

Primase and DNA polymerase act upon this extended region, creating a telomere cap that protects against loss of coding DNA from the lagging strand during multiple replications.

Thus eukaryotic DNA replication ends with two DNA molecules, each with a parental and newly-synthesized strand, numerous origins of replication, and telomeres.